Agent for potentiating the effect of interleukin-8

ABSTRACT

The inventors provide an agent for potentiating the effect of interleukin-8, which comprises dehydroepiandrosterone sulfate or a pharmaceutically acceptable salt thereof as an active ingredient. An agent for potentiating the effect of IL-8 of the present invention potentiates the effect of IL-8 by activating the receptor of IL-8 and increasing the number of receptors.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority based on PCT Application No.PCT/JP96/01774 filed Jun. 27, 1996 identifying the United States as anelected country.

TECHNICAL FIELD

This invention relates to an agent for potentiating the effect ofinterleukin-8. In detail, the invention relates to an agent, whichcomprises dehydroepiandrosterone sulfate or a pharmaceuticallyacceptable salt thereof as an active ingredient, for potentiating theeffect of interleukin-8.

BACKGROUND ART

Interleukin-8 (hereinafter referred to as IL-8) is a cytokine induced bymonocytes, macrophages, fibroblasts, vascular endothelial cells, skinkeratinized cells, renal mesangial cells, epithelial cells of intestineand respiratory tract, liver parenchyma cells and various tumor cells,and is known as a potent chemotactic factor affecting neutrophil,T-lymphocyte and basophil.

IL-8 has been reported to effectively influence the maturity of theuterine cervix in delivery and interrupted pregnancy (see WO93/09796),to have a therapeutic effect on allergic disease causing asthma, etc. byinhibiting histamine—releasing factor (HRF) from basophils (seeWO92/01465), to have a therapeutic effect on Alzheimer disease andHuntington disease etc. by protective effects on the neuronal cell (seeChemical Abstracts Vol.119: 109015k), to be effective as an acceleratingagent for healing wounds such as skin burns by accelerating the increasein vascular endothelial cell (see Chemical Abstracts Vol.119: 201760k)and so on. Furthermore, IL-8 is expected to develop an agent to targetimprovement of immunodeficiency, prevent opportunistic infection,promote anti-tumor effect, etc. by activating neutrophils and enhancinganti-bacterial effects [Zoketsu Inshi (Hematopoietic Factor), Vol.2,No.1, 46, 1991 and Med. Immunol., Vol.20, No.3, 305, 1990].

Dehydroepiandrosterone sulfate (hereinafter referred to as DHAS) or apharmaceutically acceptable salt thereof improve the maturity of theuterine cervix and the sensitivity of uterine musculature to oxytocin inthe late phase of pregnancy (see U.S. Pat. No. 4,005,200). The sodiumsalt performed well as an agent to improve the maturity of the uterinecervix. It is also well known that DHAS or a pharmaceutically acceptablesalt thereof are effective as therapy for dementia (see U.S. Pat. No.4,868,161), therapy and prevention of hyperlipemia (see GB2208473),therapy for osteoporosis (see U.S. Pat. No. 5,116,828) and therapy forulcer (see Chemical Abstracts Vol. 122 142528q).

Furthermore, DHAS or a pharmaceutically acceptable salt thereof havebeen reported to increase the production of interleukin-2 andinterleukin-4 (see WO91/04030), normalize the level of interleukin-6induced abnormally by injury, aging and autoimmune disease (seeWO93/21771).

However, neither DHAS or a pharmaceutically acceptable salt thereof hasbeen reported to increase the effect of IL-8.

DISCLOSURE OF THE INVENTION

The inventors have now completed an invention by which DHAS or apharmaceutically acceptable salt thereof increase the effect of IL-8 byactivating both receptor, type I and type II of IL-8 receptor, andfurther by increasing the number of receptors.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph showing concentrations of IL-8 in the culturesupernatant of DHAS sodium salt•dihydrate (hereinafter referred to asDHAS•Na)-treated group [0.01 mg, 0.1 mg, 1 mg as DHAS sodium salt(anhydride)] and the control group.

FIG. 2 is a graph showing the number of neutrophils in the uterinecervix in DHAS•Na-treated, IL-8 -treated, DHAS•Na and IL-8-treated andcontrol groups.

FIG. 3 is a graph showing concentrations of collagen, which weredemonstrated as the optical density, in the uterine cervix in DHAS•Na—,IL-8- and DHAS•Na and IL-8-treated groups.

BEST MODE FOR PRACTICING THE INVENTION

A pharmaceutically acceptable salt of DHAS used in present invention canbe for example a salt or the anhydride of alkaline metals such assodium, potassium, etc., or a salt of organic amine such as arginine,ethanol amine, etc..

In IL-8 therapy for various diseases, for example therapy to promote thematurity of the uterine cervix in the late phase of the pregnancy, andto treat senile dementia, Alzheimer disease, Parkinson disease,Huntington disease, diabetic neuropathy, immunodeficiency, opportunisticinfection, skin burn, wound, angiopathy, etc. the agent, which comprisesDHAS or its pharmaceutically acceptable salts as an active ingredient,for potentiating the effect of IL-8 in present invention accelerates thetherapeutic effect of IL-8 on the above diseases in combination withIL-8.

The agent for potentiating the effect of IL-8 in present invention isusually used for oral or parenteral administration to humans.

As the dosage form for oral administration, tablets, granules, finegranules, powders, etc. are mentioned. These dosage forms can beprepared by the conventional procedure by adequately mixing DHAS or apharmaceutically acceptable salt thereof and conventional phramaceuticaladditives such as lactose, corn starch, crystalcellulose, magnesiumstearate, hydroxypropylcellulose, talc, etc.

As the dosage form for parenteral administration, intravenous injectionsand suppositories are mentioned. Intravenous injections are, forexample, the lyophilized form to be extemporaneously dissolved, preparedby adequately dissolving DHAS or a pharmaceutically acceptable saltthereof and stabilizers (e.g. neutral or basic amino acids, etc. such asglycine, alanine, leucine, arginine, histidine, etc. ) in purified waterand freeze-drying in a conventional procedure. Suppositories areprepared, for example, by casting into molds and then cooling the meltedsuppository base and DHAS or a pharmaceutically acceptable salt thereof,stabilizers and absorption enhancers (e.g. neutral or basic amino acids,such as glycine, alanine, leucine, arginine, histidine, etc., orhydroxycarboxylic acid such as citric acid, L-tartaric acid), forexample after they were dispersed in hard fats having a hydroxy value ofnot more than 50, and so on.

IL-8 in combination with an agent for potentiating the effect of IL-8 inpresent invention was usually administered parenterally to humans.

As the dosage form for parenteral administration of IL-8, intravenousinjections, suppositories and ointments are mentioned. Intravenousinjections are prepared in a conventional procedure by adequately mixingIL-8 with conventional pharmaceutical additives and then by dissolvingin purified water or saline and so on. Suppositories are prepared, forexample by casting into molds and then cooling the melted suppositorybase along with IL-8 and, if necessary, pharamaceutical additives forsuppositories together, for example after they were dispersed in hardfats. Ointments are prepared in a conventional procedure by melting ordispersing IL-8 into the ointment base.

The dose of the agent for potentiating the effect of IL-8 in presentinvention may vary depending on the route of administration, theserverity of the symptom, the age of patient, the body weight, etc. butis usually administerd 0.5 to 100 mg/kg of DHAS per day in adults in asingle dose or in the multiple doses divided in 2 or 3 doses with IL-8at the same time, or immediately before or after the IL-8administration.

The dose of IL-8 may vary depending on the route of administration, theserverity of the symptom, the age of patient, the body weight, etc. butis usually administerd 0.1 ng/kg to 10 mg/kg per day in adults in asingle dose or in the multiple doses divided in 2 or 3 doses.

Furthermore, in the case of simultaneously administering an agent forpotentiating the effect of IL-8 in present invention and IL-8, it isalso possible to administer by preparing mixed preparations of DHAS or apharmaceutically acceptable salt thereof and IL-8.

Potentiating the effect of IL-8 by DHAS or its pharmaceuticallyacceptable salts was evaluated using uterine cervical tissues. As aresult, DHAS or a pharmaceutically acceptable salt thereof activated thereceptor of IL-8 and also increased the number of receptors (seeExperiment 1 as below). It was proven that they accelerated collagenaseactivity, elastase activity and gelatinase activity, consequentlyaccelerated the degradation of collagen. Furthermore, these effects weremarkedly accelerated by a combination of DHAS or a pharmaceuticallyacceptable salt thereof and IL-8 (see Experiment 2 as below).

The effests of the agent of the present invention are illustrated by thefollowing Experiments.

EXPERIMENT 1

1. Test sample

DHAS•Na

2. Experimental procedure

Biopsy specimen of uterine cervices obtained during cesarean section ofpregnant women (n=8, 38 to 40 weeks of pregnancy) were dissected afteraccepting the agreement of the patient, divided into 2, and thenincubated for 24 hours in minimum essential medium (MEM) under 37° C.and 5% CO₂. Half of the incubated biopsy specimens was further incubatedin MEM dissolved drugs [DHAS•Na (anhydride) 0.01 mg, 0.1 mg, 1 mg], andprepared as samples for the measurement of IL-8 concentrations(hereinafter referred to as DHAS•Na-treated group). The other half ofthe biopsy specimen was further prepared as controls (hereinafterreferred to as control group) by continuing incubation for 24 hours.IL-8 concentrations in the culture supernatant of DHAS•Na-treated andcontrol groups were measured using a radio-immuno assay method.

Each frozen preparation from 5 random fields of the tissue of biopsyspecimen in DHAS•Na-treated and control groups were stainedimmunohistologically for IL-8 receptor type I and type II using aStreptavidin—biotin complex—peroxidase kit (Dako, USA). Anti-IL-8receptor type I and type II antibody was prepared using rabbitsaccording to a method of Morohashi et al. (Journal of Leukocyte Biology,57, 180-187, 1995). Analysis was performed by measuring the opticaldensity and the mean was calculated. Using an image-analyzer,histological analysis was performed using a microscope connected to avideo-camera.

Furthermore, the cervical tissues of DHAS•Na-treated and control groupswere incubated in the medium containing IL-8-FITC complex 100 μl for 2hours in a dark room, and then frozen preparations were prepared forobservation under the fluorescence microscope.

3. Results

The results are shown in FIG. 1.

FIG. 1 is the results of IL-8 concentrations of the culture supernatantin the cervical tissues. As clearly shown in FIG. 1, IL-8 concentrationsof the culture supernatant in DHAS•Na-treated group were significantlydecreased compared to those in the control group. IL-8 concentrationsdecreased in proportion to the increase in DHAS•Na concentrations.

IL-8 receptor type I and type II incubated with DHAS•Na was morestrongly stained than that in the control group. The fluorescence inDHAS•Na-treated group incubated with IL-8-FITC was markedly higher thanthat in the control group, and increased in a dose-dependent manner.

Taken together, DHAS or its pharmaceutically acceptable salts activateIL-8 receptor and increase the number of receptors, consequentlyincrease binding to IL-8 receptor and decreasing IL-8 concentrations inthe culture supernatant.

EXPERIMENT 2

1. Test samples (according to Preparations 1 to 4 as follows, samplesfor tests were prepared)

a) the virginal suppositories containing of DHAS•Na (anhydride) 10 mg(DHAS•Na-treated group)

b ) the virginal suppositories containing of human recombinant IL-8 100ng (IL-8-treated group)

c) the virginal suppositories containing of DHAS•Na (anhydride) 10 mgand human recombinant IL-8 100 ng (DHAS•Na+IL-8-treated group)

d) the virginal suppositories of placebo (control group)

2. Experimental procedure

Sixteen primigravida rabbits (day 23 of pregnancy) were divided into 4groups, and each group of 4 rabbits was administered the test sample inthe vagina once daily for 4 days. The cervices were dissected 24 hoursafter the administration of last suppository. Cervices were examined forconsistency and dilation using Hegar's dilators. Histological sectionswere prepared to study the collagen content and neutrophil invasion ofthe cervical tissue. The collagen concentration was assessed by stainingwith picrosirius red (Sirius red F3BA Chroma-Gesell Schaft Schmid GmbHCo.) and validated as a histological method of determining thepolymerized collagen concentration in the cervical tissues. Histologicalanalysis was performed by measuring the optical density from 5 randomfields of the tissue of each biopsy specimen and the mean opticaldensity was calculated. In picrosirus red staining, the greater thecollagen concentration, the greater the birefringence, and hence thegreater the percentage of light transmission.

Histological sections were stained immunohistologically for surfaceantigen RT2 that is found in rabbit neutophils using anti-rabbit RT2monoclonal antibody (Collagenase type I activity measurement, Yagai Co.,Japan). The number of neutrophils (magnification×20, but avoiding cellsthat were within blood vessels) in 5 random fields of the tissue of eachbiopsy specimen was counted, and the mean was calculated

From each rabbit cervix 100 mg of tissue was cut, homogenized withphosphate buffered saline, and the supernatant fluid was collected.Collagenase activity in the supernatant was measured using a specificchromogenic substrate for granulocyte elastase S-2484(L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, KABI Diagnostic,Sweden). Gelatinase activity was measured using a special kit(Gelatinase activity measurement, Yagai Co. Japan).

3. Results

The results are shown in FIG. 2, FIG. 3 and Table 1.

FIG. 2 shows the number of neutrophils. Table 1 summarizes enzymeactivities in each treated group. FIG. 3 shows the collagenconcentrations.

TABLE 1 Enzyme activity (mean ± SD, U/100 mg) Groups/ DHAS · Na EnzymeControl DHAS · Na IL − 8 + IL − 8 Collagenase 0.55 ± 0.13 0.82 ± 0.220.92 ± 0.18 1.50 ± 0.18 Elastase 5.75 ± 0.96 9.60 ± 0.28 12.0 ± 1.1 14.9 ± 1.3  Gelatinase 7.0 ± 0.8 14.0 ± 0.8  17.0 ± 0.8  20.3 ± 1.3 

As clearly shown in FIG. 2, neutrophils were significantly increased inthe DHAS•Na-treated group compared to those in the control group. Thenumber of neutrophils in the DHAS•Na+IL-8-treated group was furtherincreased.

As clearly shown in Table 1, in collagenolytic enzymes (collagenase,elastase and gelatinase), DHAS•Na-treated group showed increased enzymeactivities. DHAS•Na+IL-8-treated group showed further increases inenzyme activities.

As clearly shown in FIG. 3, the DHAS•Na-treated group showed decreasedthe collagen concentration, DHAS•Na+IL-8-treated group showed furtherdecreases in the collagen concentration. Also, cervices in theDHAS•Na+IL-8-treated group were markedly soft and dilatable compared tothose in the control group.

Taken together, the potentiating effect of IL-8 by DHAS or itspharmaceutically acceptable salts was evaluated using cervical tissue.Consequently, agents for potentiating the effect of IL-8 in thisinvention potentiated the production of neutrophils which induced therelease of collagenolytic enzymes (collagenase, elastase and gelatinase)and decreased the collagen concentration, as described above byactivating IL-8 receptor, increasing the number of receptors and thenaccelerating the binding of IL-8 to IL-8 receptor. As a result, theagents have been proved to accelerate the maturity of the uterine cervixcontaining collagen fiber. The effect was markedly accelerated by acombination of DHAS or its pharmaceutically acceptable salts and IL-8.

The present invention is illustrated in more detail by the followingExamples and Preparations.

EXAMPLE 1 Production of tablets

[Formulation]

Ingredient Amount (g) Sodium salt of DHAS · dihydrates 109.2 Lactose64.8 Corn starch 31.6 Hydroxypropylcellulose 1.8 Magnesium stearete 0.6

[Procedure]

Sodium salt of DHAS•dihydrates, lactose and corn starch werehomogeneously mixed up and the mixture was passed through a 60 meshsieve. An aqueous solution of hydroxypropylcellulose was added to thesieved mixture, followed by kneading, and drying. Then, magnesiumstearate was added and the whole mixture was tableted to give tabletseach weighing 208 mg. Thus, tablets each containing 100 mg of Sodiumsalt of DHAS (on the anhydrate basis) are prepared.

EXAMPLE 2 Production of an injection

[Formulation]

Ingredient Amount (g) Sodium salt of DHAS · dihydrates 43.7 Glycine 40.0

[Procedure]

Purified water was added to glycine for dissolution of the latter.Thereto was added sodium salt of DHAS•dihydrates with warming to causedissolution of the same. Then, the whole amount was made 2000 ml. Theresultant solution was sterilized by filtration and then distributed in5 ml portions into receptacles for injection (vials), followed bylyophilization. Thus was prepared injectable preparation to beextemporaneously dissolved, each vial containing 100 mg of DHAS•Na (onthe anhydrous basis).

EXAMPLE 3 Production of virginal suppositories

[Formulation]

Ingredient Amount (g) Sodium salt of DHAS · dihydrates 43.7 Glycine 40.0Hard fat 266.3

[Procedure]

Hard fat (Witepsol™ H-15, product of Huls AG) was placed in a stainlesssteel beaker and melted by warming at 40 to 55° C. Thereto were addedsodium salt of DHAS•dihydrates and glycine, and the mixture was stirreduntil it became homogeneous. This mixture, while maintained at 37 to 55°C., was casted in 1.6 g poritions into spindle-shaped molds and thencooled to give vaginal suppositories each containing 200 mg of DHAS (onthe anhydrous basis).

EXAMPLE 4 Production of virginal suppositories

[Formulation]

Ingredient Amount (g) Sodium salt of DHAS · dihydrates 43.7 humanrecombinant IL - 8 0.0004 Glycine 10.0 Hard fat 266.3

[Procedure]

Hard fat (Witepsol™ H-15, product of Huls AG) was placed in a stainlesssteel beaker and melted by warming at 40 to 55° C. Thereto were addedsodium salt of DHAS•dihydrates, human recombinant IL-8 and glycine, andthe mixture was stirred until it became homogeneous. This mixture, whilemaintained at 37 to 55° C., was casted in 1.6 g poritions intospindle-shaped molds and then cooled to give virginal suppositories eachcontaining 200 mg of DHAS (on the anhydrous basis) and 2 μg of humanrecombinant IL-8.

Preparation 1 [Production of virginal suppositories containing 10 mg ofDHAS (anhydrate) for adminstrating animals]

[Formulation]

Ingredient Amount Sodium salt of DHAS (on the anhydrous 100 mg basis)Glycine 100 mg Hard fat  2 ml

[Procedure]

Hard fat (Witepsol™ H-15, product of Huls AG) was placed in a stainlesssteel beaker and melted by warming at 50° C. Thereto were added sodiumsalt of DHAS (on the anhydrous basis) and glycine, and the mixture wasstirred until it became homogeneous. This mixture, while maintained at37 to 50° C., was casted in 0.2 ml poritions into molds and then cooledto give virginal suppositories each containing 10 mg of DHAS (on theanhydrous basis).

Preparation 2 [Production of virginal suppositories containing 100 ng ofhuman recombinant IL-8 for adminstrating animals]

[Formulation]

Ingredient Amount human recombinant IL - 8 1 μ g Hard fat 2 ml

[Procedure]

Hard fat (Witepsol™ H-15, product of Huls AG) was placed in a stainlesssteel beaker and melted by warming at 50° C. Thereto were added humanrecombinant IL-8, and the mixture was stirred until it becamehomogeneous. This mixture, while maintained at 37 to 50° C., was castedin 0.2 ml poritions into molds and then cooled to give virginalsuppositories each containing 100 ng of human recombinant IL-8.

Preparation 3 [Production of virginal suppositories containing 10 mg ofDHAS (anhydrate) and 100 ng of human recombinant IL-8 for adminstratinganimals]

[Formulation]

Ingredient Amount Sodium salt of DHAS (on the anhydrous 100 mg basis)human recombinant IL - 8  1 μ g Glycine 100 mg Hard fat  2 ml

[Procedure]

Hard fat (Witepsol™ H-15, product of Huls AG) was placed in a stainlesssteel beaker and melted by warming at 50° C. Thereto were added sodiumsalt of DHAS (on the anhydrous basis), human recombinant IL-8 andglycine, and the mixture was stirred until it became homogeneous. Thismixture, while maintained at 37 to 50° C., was casted in 0.2 mlporitions into molds and then cooled to give virginal suppositories eachcontaining 10 mg of DHAS (on the anhydrous basis) and 100 ng of humanrecombinant IL-8.

Preparation 4 [Production of placebo virginal suppositories foradminstrating animals]

[Formulation]

Ingredient Amount Hard fat 2 ml

[Procedure]

Hard fat (Witepsol™ H-15, product of Huls AG) was placed in a stainlesssteel beaker and melted by warming at 50° C. This one, while maintainedat 37 to 50° C., was casted in 0.2 ml poritions into molds and thencooled to give placebo virginal suppositories.

What is claimed is:
 1. A method of therapy using IL-8 to promotematurity of the uterine cervix in late phase pregnancy, comprising thesteps of: (a) administering an amount of IL-8 to a subject in need ofsaid therapy; and (b) administering to the subjectdehydroepiandrosterone sulfate (DHAS) or a pharmaceutically acceptablesalt thereof in an amount that potentiates the therapeutic effect ofIL-8, thereby promoting maturity of the uterine cervix in late phasepregnancy in the subject.
 2. The method of claim 1, wherein said IL-8therapy increases activity of collagenolytic enzymes.
 3. The method ofclaim 2, wherein said collagenolytic enzymes are collagenase, elastaseor gelatinase.
 4. The method of claim 1, wherein the subject is human.